Recently, a number of biosensors for the early diagnosis and rapid prognosis of human diseases (e.g., acute myocardial infarction, cancer, infectious diseases) have been developed. This is because various fatal human diseases can often be treated if they are diagnosed early. For example, National Cancer Institute (NCI) noticed that 5-year survival rate of breast cancer patients from 2004 to 2010 in USA was 89.2%. In addition, American Cancer Society recently reported that 5-year survival rate of breast cancer patients in stages 0 and 1 is 100%. Also, 93% of breast cancer patients in stages 2 survive for 5 years or more.
In order to early diagnose human diseases, most of biosensors have generally been developed based on the principle of immunoassay operated with specific antibodies (e.g., capture and detection antibodies) capable of rapidly binding a biomarker existing in human samples (e.g., whole blood, serum, plasma, urine). A detection antibody conjugated with a specific enzyme such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP) is widely used to enhance the sensitivity of biosensor. Sandwich enzyme immunoassay with a capture antibody and a detection antibody-conjugated HRP or ALP is highly sensitive with acceptable accuracy and reproducibility. However, antibodies and enzymes obtained from the sacrifice of animals are very expensive and intractable in ambient condition.
Many research groups have developed various methods to immobilize multiple detection antibodies and enzymes on the surface of nanoparticles such as gold, platinum, and silver. Using the nanoparticles modified with detection antibody-conjugated HRP, it was possible to develop more sensitive enzyme immunoassays. However, the system of biosensor was more expensive and more complicated than conventional enzyme immunoassay.
Since 1990, various aptamers composed of DNA, RNA, or peptide have been discovered and designed with the systematic evolution of ligands by exponential enrichment (SELEX). This is because the function of cost-effective aptamers are similar to or better than that of expensive antibodies. For example, the binding rate of a specific aptamer to capture a biomarker in human sample is similar to or more rapid than that of a conventional antibody.
Using the advantages of aptamer, many biosensors were developed as a medical device because the selectivity of biosensor using DNA or RNA aptamer conjugated with fluorescence dye (e.g., fluorescein, 6-FAM) are acceptable for the early diagnosis of human diseases. In general, the biosensor devised using an aptamer is called the aptasensor. Unfortunately, the sensitivity of aptasensor using a detection aptamer-conjugated fluorescent dye was not as good as that of biosensor using a detection antibody conjugated with HRP. In order to solve the disadvantages of aptasensor, recently, various nanoparticles as well as microspheres (e.g., magnetic beads) were used to enhance the sensitivity of aptasensor. Thus, the complicated aptasensors, like enzyme immunoassays, were operated with time-consuming procedures such as multiple incubations and washings.
Hemin (FIG. 1(a)) is known as a HRP-mimicking compound. Also, the efficiency of hemin bound with DNA hemin aptamers, called HRP-mimicking G-quadruplex DNAzyme (FIG. 1(b)), is as good as that of HRP in enzyme immunoassay (Whillner, I., Shlyahovsky, B., Zayats, M., and Willner, B. (2008) DNAzymes for sensing, nanobiotechnology and logic gate applications, Chem. Soc. Rev. 37, 1153-1165). Also, the HRP-mimicking G-quadruplex DNAzyme is stable in ambient condition even though the expensive HRP must be stored in a freezer (<−20° C.). Recently, several aptasensors using a mixture of aptamers and HRP-mimicking G-quadruplex DNAzyme have been developed as diagnostic methods. The aptasensor using HRP-mimicking G-quadruplex DNAzyme was more sensitive than the aptasensor using fluorescent dye. However, using a simple mixture of aptamers and HRP-mimicking G-quadruplex DNAzyme requires time-consuming procedures such as multiple incubations, as well as multiple washings after each incubation.
Colorimetric, fluorescence, and chemiluminescence are widely applied as optical detection methods of conventional immunoassays as well as aptasensors. Chemiluminescence detection is more sensitive than other optical sensors because the background of chemiluminescence emitted from a chemical reaction is lower than those of absorbance and fluorescence generated by a light source such as halogen, Xenon, and laser generated with high-voltage power supply. Luminol chemiluminescence is widely applied as a detection method of enzyme immunoassay using HRP and aptasensor using G-quadruplex DNAzyme even though 1,1′-oxalyldiimidazole (ODI) chemiluminescence is more sensitive than luminol chemiluminescence in enzyme immunoassay. ODI chemiluminescence has never been used as a detection method of aptasensor using G-quadruplex DNAzyme yet.
Carcinoembryonic antigen (CEA) is well-known as a breast cancer marker. Currently, sandwich enzyme immunoassay using a detection antibody conjugated with HRP or ALP is widely applied. Recently, two different types of DNA aptamers, capable of rapidly capturing CEA in a sample, have been developed. However, a highly sensitive aptasensor, using the CEA aptamer, has yet to be developed for the early diagnosis of breast cancer.